Beneficial uses of microorganisms are well known in the art and have been documented at great length. Many patents have issued which claim new microbial processes pertaining to the production of antibiotics, enzymes, ethanol, and a multitude of other useful products. Microorganisms are also used to clean up toxic wastes and oil spills, mill pests, recover minerals, and provide nutrients to plants. It has been known for many years that some organisms produce compounds which are toxic to other organisms. The production of the antimicrobial compound penicillin by penicillium mold is one such example.
Microorganisms are particularly attractive candidates for use in making and delivering organic compounds because they can be extremely efficient and safe. The modern tools of genetic engineering have greatly enhanced the ability to exploit the efficiency and relative safety of microbes. Even in the absence of genetic manipulation, however, microbes can perform highly specific tasks which make them indispensable in certain applications. Thus, there is a constant ongoing search in many areas of research for new microbes with specific advantageous properties. The subject invention concerns the discovery of one such microbe.
The tree species Melaleuca quinquenervia (Cam.) Blake (Melaleuca) is an exotic pest species which is native to Australia and was introduced into Florida in the early 1900's as an ornamental tree and possibly as a commercial source of wood. Several of Melaleuca's innate characteristics have facilitated its spread throughout South Florida. Melaleuca grows more densely in Florida than in Australia and "crowds out" native plants. Prolific seed production, fire adaptation and release from natural competition, insect feeding and disease further abet its competitive ability. The Melaleuca may become a large tree exceeding 50 feet in height. The tree may have a single trunk or have multiple stems arising from the base of the tree. The bark covering the trunk is white to cream-colored and is very thick and soft, and easily peels in multiple layers from the tree. The tree is easily recognized when flowering, being covered with clusters of white flowers born on the ends of the twigs. Melaleuca flowers throughout the year in Florida, with heavier blooms reported during the wet season with lighter blooms occurring throughout the winter. Individual trees have been reported to bloom as many as five times a year and an individual branch may have three or more blooms each year. Seed capsules which are formed on the flower spike, are from 0.1 to 0.2 inches long and are short and cylindrical. Each capsule, which contains over 200 seeds, may remain attached to the branch for an extended period of time.
A large, mature melaleuca tree has a high reproductive potential as the branches contain millions of seeds stored in the capsules. By flowering three to five times yearly, large numbers of seedlings are produced. These seedlings can, in turn, produce seeds within two to three years, and a mature tree can store over 20 million seeds. Encroachment into ecosystems formerly devoid of Melaleuca is irreversible, permanently replacing natural plant communities and the animals that live in them. Melaleuca was planted from seeds obtained from Australia in the early 1900's at two coastal locations. The present distribution of melaleuca is predominantly centered around the areas of original introduction. Its spread was enhanced through its use as wind breaks and fence rows, and its popularity as a fast growing ornamental. Canals have most likely facilitated the spread of the buoyant seeds in to the interior of conservation areas where relatively undisturbed inland wetlands have been invaded. Sites conducive to Melaleuca development are usually poorly-drained areas which have high water table levels or are flooded periodically each year. These sites comprise much of the ecologically-sensitive wetland areas of South Florida, including the Everglades National Park, the Big Cypress Preserve, and the Loxahatchee National Wildlife Refuge.
Melaleuca is highly resistant to stress, including herbicides and fire. Not only is this species physically resistant to fire, but the seed capsules are stimulated to open by the extreme heat and drying produced by fire. The trees grow rapidly, even when completely submerged in flood waters for periods of six months or longer, and they resume vigorous growth after the water recedes.
Melaleuca has been identified as a potential threat to South Florida's water supply. Future spread of melaleuca throughout the Everglades has the potential to impact regional surface water supplies by replacing open grassy paries with forest.
A task force assigned to study the melaleuca problem has concluded:
"It is the consensus opinion of the [task force] that the uncontrolled expansion of melaleuca constitutes one of the most serious ecological threats to the biological integrity of South Florida's natural systems."
Control of this encroachment is a formidable task, even when chemical herbicides are applied either to individual trees or to groups of trees by aerial spraying. Eradication frequently requires two or three applications of herbicides which increases herbicidal contamination of wetlands. Thus, chemical weed control programs are seriously inadequate for the control of Melaleuca.
Also, the use of chemical pesticides in agriculture is currently a major concern in the U.S. For example, pesticides are being blamed for an epidemic of cancer in children and young adults in the San Joaquin Valley (Weisskopf, M. [1988] The Washington Post Weekly Edition 5(47):10-11, Washington, D.C.). New technologies in detection methods are enabling researchers to find pesticides in the environment that were previously thought to be totally degraded. Perhaps the major public concern of the 1980's is protection of groundwater. The Environmental Protection Agency (EPA) estimates that 100,000 of the nation's 1.3 million wells are contaminated with pesticides (Fleming, M. H. [1987] Amer. J. Alterative Agriculture 2:124-130). This has alarmed the general public since 50% of all Americans depend on groundwater wells for their fresh water supplies. Because herbicides are so widely used in agriculture, and because they are often applied directly to the soil, the potential for movement into groundwater by leaching is perhaps greater than any other pesticide. Other inadequacies of chemical controls include lack of residual control, injury to non-target organisms, undesirable residues in harvested products, and carryover in subsequent crops. Among the chemical herbicides now being used in efforts to control Melaleuca are Arsenal (isopropylamine salt of 2-[4,5-dihydro-4-methyl-4-91-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridi necarboxylic acid), Bonvel 720 (diethylamine salt of 2,4-dichlorophenoxy-acetic acid+dimethylamine salt of 3,6-dichloro-0-anisic acid), Garlon 3A (triethylamine salt of 3,5,6,-tricloro-2-pyridinyloxyacetic acid), Rodeo (isopropylamineamine salt of N-(phosphonomethyl)glycine), Spike (N-[5-(1,1-dimethylethyl)-1,3,4,-thiadiazol-Z-yl]-N,N'dimethylurea), and Velpar (3-cyclohexyl-6-dimethylamino) 1-methyl-1,3,5-triazine-2,4(1H,3H)-dione). Certainly, the use of chemical herbicides must be avoided or reduced to the extent possible in the environmentally sensitive wetlands of South Florida.
Therefore, the use of bioherbicides is becoming an increasingly important alternative to chemical herbicides. This importance is exemplified by several patents which have been issued for bioherbicides and their use. Some of these patents, by way of illustration, are as follows: U.S. Pat. No. 3,849,104 (control of northern jointvetch with Colletotrichum gloeosporioides Penz. aeschynomene); U.S. Pat. No. 3,999,973 (control of prickly sida [teaweed] and other weeds with Colletotrichum malvarum); U.S. Pat. No. 4,162,912 (control of milkweed vine with Araujia mosaic virus); U.S. Pat. No. 4,626,271 (Cyanobacterin Herbicide); and U.S. Pat. No. 4,915,726 (Biological Control of Dodder).
Melaleuca quinquenervia has not been reported to have any natural enemies in Florida capable of inducing mortality. Fungi of the genus Botryosphaeria, including B. ribis, have been shown to grow on other species of plants (Ramos, L. J., S. P. Lara, R. T. McMillan, Jr., K. R. Narayanan [1991] Plant Dis. 75:315-318; Venkatasubbaiah, P., T. B. Sutton, W. S. Chilton [1991] Phytopath. 81:243-247; Webb, R. S. [1983] Plant Dis. 67:108-109). However, the fungus is not shown to cause sufficient damage to induce mortality in any of the specifies shown to be infected with the fungus.
Mellein and 4-hydroxymellein are isocoumarin compounds which have previously been described (Moore, J. H., N. D. Davis, and U. L. Diener [1972] "Mellein and 4-hydroxymellein production by Aspergillus ochraceus wilhem,' Microbiology 23(6):1067-1072; Cole, R. J., J. H. Moore, N. D. Davis J. W. Kirksey, and U. L. Diener [1971] "4-hydroxymellein: A new metabolite of Aspergillus ochraceus J. Agr. Food Chem., 19(5):909). Phytotoxic properties have not previously been reported for these compounds. Nor has there been any report that these compounds are produced by Botryosphaeria ribis.